Microspectrofluorometry in conjunction with microelectrophoretic injection of metabolic intermediates is being used to follow in situ the metabolic activity of normal versus transformed or malignant cells and the intracellular distribution, interactions or metabolization of fluorescent carcinogens. Using NAD(P) yields (reversibly) NAD(P)H transients due to microinjection of glucose-6-P plus allosteric activators, the rate constants of NAD(P) reduction and reoxidation, and the relationship of overall metabolic rate to concentration of substrate introduced (So), e.g., R equals a(S)b, can be now determined in a variety of cell lines: i.e. EL2, L, NCTC 6991 normal rat fibroblasts versus NCTC 6992 transformed, NCTC 7505A normal mouse fibroblast versus NCTC 7505B transformed, CG 787 normal glia versus MG 251 glioma, HPM 73 highly melanizing melanoma versus less melanizing HPM 67. Studies on the glycolytic chain will be complemented by investigation of other pathways, e.g., pentose shunt, gluconeogenesis, mitochondrial-cytosolic interactions. The findings will be correlated to phases of cell cycle and intracellular structure. The same approach will be extended to cell lines such as the clone C3H/10 T1/2CL8 successfully used for the qualitative and quantitative studies of oncogenic transformation by chemicals, many of which are fluorescent. A grating microspectrofluorometer is being equipped with a long working phase and multioptional dichromatic high resolution mirror arrangement for simultaneous phase-fluorescence observations. Coenzyme fluorescence spectra with emphasis on elucidation of the flavin component will be related to metabolic activity or cell type. Preliminary spectral studies suggest that both the penetration and elimination of carcinogen molecules follow exponential laws. The in situ approach will be concentrated on the localization and detection of carcinogen interactions or metabolites, attempting to identify metabolites by their fluorescence spectra. Parallel topographic studies by multisite microfluorometry will aim at the detection of associated or concomitant changes of metabolic activity (or deregulations).